Conserved POU Binding DNA Sites in the Sox2 Upstream Enhancer Regulate Gene Expression in Embryonic and Neural Stem Cells

Department of Biotechnology and Biosciences, University of Milano-Bicocca, piazza della Scienza 2, Milano 20126, Italy.
Journal of Biological Chemistry (Impact Factor: 4.57). 11/2004; 279(40):41846-57. DOI: 10.1074/jbc.M405514200
Source: PubMed


The Sox2 transcription factor is expressed early in the stem cells of the blastocyst inner cell mass and, later, in neural stem cells. We previously identified a Sox2 5'-regulatory region directing transgene expression to the inner cell mass and, later, to neural stem cells and precursors of the forebrain. Here, we identify a core enhancer element able to specify transgene expression in forebrain neural precursors of mouse embryos, and we show that the same core element efficiently activates transcription in inner cell mass-derived embryonic stem (ES) cells. Mutation of POU factor binding sites, able to recognize the neural factors Brn1 and Brn2, shows that these sites contribute to transgene activity in neural cells. The same sites are also essential for activity in ES cells, where they bind different members of the POU family, including Oct4, as shown by gel shift assays and chromatin immunoprecipitation with anti-Oct4 antibodies. Our findings indicate a role for the same POU binding motifs in Sox2 transgene regulation in both ES and neural precursor cells. Oct4 might play a role in the regulation of Sox2 in ES (inner cell mass) cells and, possibly, at the transition between inner cell mass and neural cells, before recruitment of neural POU factors such as Brn1 and Brn2.

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    • "Moreover, we tried to ameliorate NPC-like outputs of our " FPd " procedure by super-imposing to it select gene manipulations, supposed to help mimicking the dynamic transcriptional milieu of the early pallial field (Bell et al., 2001; Uchikawa et al., 2011). However, neither delayed overexpression of Emx2 and Lhx2, nor early overexpression of two key factors sustaining NSC programmes (Catena et al., 2004; Iwafuchi-Doi et al., 2011; Tanaka et al., 2004), Sox2 and Brn2, was successful (Fig. S2A,B). Finally, we tried to facilitate reprogramming by counteracting the heat shock machinery, which can buffer phenotypic consequences of genetic variation and ease inheriting of epigenetic traits (Ruden, 2011; Taipale et al., 2010). "
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    ABSTRACT: In this study, we assayed the capability of four genes implicated in embryonic specification of the cortico-cerebral field, Foxg1, Pax6, Emx2 and Lhx2, to reprogram mouse embryonic fibroblasts toward neural identities. Lentivirus-mediated, TetON-dependent overexpression of Pax6 and Foxg1 transgenes specifically activated the neural stem cell (NSC) reporter Sox1-EGFP in a substantial fraction of engineered cells. The efficiency of this process was enhanced up to ten times by simultaneous inactivation of Trp53 and co-administration of a specific drug mix inhibiting HDACs, H3K27-HMTase and H3K4m2-demethylase. Remarkably, a fraction of the reprogrammed population expressed other NSC markers and retained its new identity, even after switching off the reprogramming transgenes. When transferred into a pro-differentiative environment, Pax6/Foxg1-overexpressing cells activated the neuronal marker Tau-EGFP. Frequency of Tau-EGFP positive cells was almost doubled upon delayed delivery of Emx2 and Lhx2 transgenes. A further improvement of the neuron-like cells output was achieved by inhibition of the BMP and TGFβ pathways. Tau-EGFP positive cells were able to generate action potentials upon injection of depolarizing current pulses, further indicating their neuron-like phenotype.
    Molecular and Cellular Neuroscience 10/2013; 57. DOI:10.1016/j.mcn.2013.10.004 · 3.84 Impact Factor
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    • " Shushan et al . , 1998 ) . There are also some evidences supporting the existence of compos - ite elements on the regulatory regions of genes involved in hESC . POU5F1 can bind to an octamer element , ATGCAAAT , on the regulatory regions of Fgf4 , Utf1 , Opn , Rex1 / Zpf42 , Fbx15 and SOX2 ( Ben - Shushan et al . , 1998 ; Botquin et al . , 1998 ; Catena et al . , 2004 ; Dailey et al . , 1994 ; Nishimoto et al . , 1999 ; Tokuzawa et al . , 2003 ; Tomioka et al . , 2002 ; Yuan et al . , 1995 ) . These sites have been demonstrated to be cru - cial for transcriptional activity of their genes . The octamer elements within the enhancers of Fgf4 , Utf1 , Opn , Fbx15 and SOX2 are close to Sox2 - binding sox "
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    ABSTRACT: Self-proliferation and differentiation into distinct cell types have been made stem cell as a promising target for regenerative medicine. Several key genes can regulate self-renewal and pluripotency of embryonic stem cells (hESCs). They work together and build a transcriptional hierarchy. Coexpression and coregulation of genes control by common regulatory elements on the promoter regions. Consequently, distinct organization and combination of transcription factor binding sites (TFBSs modules) on promoter regions, in view of order and distance, leads to a common specific expression pattern within a set of genes. To gain insights into transcriptional regulation of hESCs, we selected promoter regions of eleven common expressed hESC genes including SOX2, LIN28, STAT3, NANOG, LEFTB, TDGF1, POU5F1, FOXD3, TERF1, REX1 and GDF3 to predict activating regulatory modules on promoters and discover key corresponding transcription factors. Then, promoter regions in human genome were explored for modules and 328 genes containing the same modules were detected. Using microarray data, we verified that 102 of 328 genes commonly upregulate in hESCs. Also, using output data of DNA-Protein interaction assays, we found that 42 of all predicted genes are targets of SOX2, NANOG and POU5F1 . Additionally, a protein interaction network of hESC genes was constructed based on biological processes and interestingly, 126 downregulated genes along with upregulated ones identified by promoter analysis were predicted in the network. Based on the results, we suggest that the identified genes, coregulating with common hESC genes, represent a novel approach for gene discovery based on whole genome promoter analysis irrespective of gene expression. Altogether, promoter profiling can be used to expand hESC transcriptional regulatory circuitry by analysis of shared functional sequences between genes. This approach provides a clear image on underlying regulatory mechanism of gene expression profile and offers a novel approach in designing gene networks of stem cell.
    Gene 09/2013; 531(2). DOI:10.1016/j.gene.2013.09.011 · 2.14 Impact Factor
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    • "Concretely, set 1 was selected by the review of published articles and included Nanog (Mitsui et al., 2003; Loh et al., 2006), Pou5f1 (Okazawa et al., 1991; Catena et al., 2004; Akamatsu et al., 2009), Zfl42 (Shi et al., 2006; Scotland et al., 2009), Fgfr1 (Jukkola et al., 2006; Yang et al., 2008; Lee et al., 2009), Sox2 (Tomioka et al., 2002; Graham et al., 2003; Tanaka et al., 2004; Jin et al., 2009), and Oligo2 (Ahn et al., 2008). RARs were also added to set 1 to assess the effects of RA. "
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    ABSTRACT: We have previously established a protocol for the neural differentiation of mouse embryonic stem cells (mESCs) as an efficient tool to evaluate the neurodevelopmental toxicity of environmental chemicals. Here, we described a multivariate bioinformatic approach to identify the stage-specific gene sets associated with neural differentiation of mESCs. We exposed mESCs (B6G-2 cells) to 10(-8) or 10(-7) M of retinoic acid (RA) for 4 days during embryoid body formation and then performed morphological analysis on day of differentiation (DoD) 8 and 36, or genomic microarray analysis on DoD 0, 2, 8, and 36. Three gene sets, namely a literature-based gene set (set 1), an analysis-based gene set (set 2) using self-organizing map and principal component analysis, and an enrichment gene set (set 3), were selected by the combined use of knowledge from literatures and gene information selected from the microarray data. A gene network analysis for each gene set was then performed using Bayesian statistics to identify stage-specific gene expression signatures in response to RA during mESC neural differentiation. Our results showed that RA significantly increased the size of neurosphere, neuronal cells, and glial cells on DoD 36. In addition, the gene network analysis showed that glial fibrillary acidic protein, a neural marker, remarkably up-regulates the other genes in gene set 1 and 3, and Gbx2, a neural development marker, significantly up-regulates the other genes in gene set 2 on DoD 36 in the presence of RA. These findings suggest that our protocol for identification of developmental stage-specific gene expression and interaction is a useful method for the screening of environmental chemical toxicity during neurodevelopmental periods.
    Frontiers in Genetics 08/2012; 3:141. DOI:10.3389/fgene.2012.00141
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